Treatment of hormone dependent breast cancer induces regressions lasting 12-18 months in women but relapses invariably occur. Additional responses to subsequent hormonal therapies may occur but reversion later to a hormone independent state ultimately results. We postulate that these sequential responses reflect adaptive changes in tumors whereby hypersensitivity to both the proliferative and pro-apoptotic effects of estradiol occurs in response to up-regulation of growth factor pathways. The current proposal examines why growth factor pathway up-regulation occurs and what mechanistic changes ensue. Our recent data suggest that enhanced non-genomic effects of estradiol acting at the level of the cell membrane might be responsible. Membrane related estrogen receptors co-opt growth factor pathways and utilize the IGF-1 and EGF receptors as well as Shc, Grb-2, and SOS to stimulate MAP kinase, PI-3-kinase and cell motility. In this proposal, we will pursue four Specific Aims #1: confirm the hypothesis that the non-genomic actions of ER alpha are responsible for up-regulation of the MAP kinase and PI-3 kinase pathways in adapted breast cancer cells. We will first validate three direct methods for distinguishing non-genomic effects of estradiol from those which directly stimulate gene transcription: (1) use of a membrane impermeable biotinylated estradiol (2) use of a novel knock out/rescue technique, and (3) use of a dominant negative construct directly exclusively against membrane ERalpha. We will utilize these methods to examine the role of non-genomic actions in hypersensitivity and compare the degree of utilization of non-genomic pathways in adapted and nonadapted cell lines. #2 Determine the mechanisms causing hypersensitivity to estradiol in adapted cells with respect to apoptosis. We will test the hypothesis that estradiol triggers apoptosis predominately through non-genomic estrogen receptoralpha effects by activating the stress kinase (JNK), p38 kinase and the death receptor Fas/FasL pathways as well as the Type II apoptotic, mitochondrial pathways. The strategies to establish causality include the induction of hypersensitivity to estradiol in non-adapted cells, and reversion of hypersensitivity in adapted cells using dominant negative and siRNA techniques. #3 Role of the membrane ER in dynamic membrane changes in adapted cells. We will test the hypothesis that non-genomic effects of estradiol act through the Rac/PAK1 pathways to enhance the formation of membrane changes to increase motility and invasiveness of breast cancer cells. Our working model includes a nodal point consisting of an EGF-R/FAK complex;input through non-genomic ER related pathways and output through PI-3-kinase, c-SRC and p130CAS to Rac/PAK1. #4 Effect of Blockade of mTOR on apoptosis. Determination of the mechanisms involved in apoptosis allows development of new experimental strategies for enhancement of estradiol induced apoptosis. FTS, a novel mTOR inhibitor will be used in vitro to activate the stress kinase pathway and apoptosis. In vivo, FTS will be used to enhance estradiol induced apoptosis in nude mice.